The present invention relates generally to in vitro biosynthetic systems and products thereof and more specifically to the net biosynthesis, in vitro, of chlorophyll and grana.
The prime source of energy in the biosphere is the light absorbed by chlorophyll-containing plant cells. This energy is used to fix carbon dioxide into carbohydrates by the following general formula. EQU 6 CO.sub.2 + 6 H.sub.2 O .sup.light C.sub.6 H.sub.12 O.sub.6 + 6 O.sub.2
The photosynthetic apparatus of plant and algal cells is lodged within especially organized structures called chloroplasts, which are bodies of varied shape, 3 to 10 .mu. long and 0.5 to 2 .mu. in diameter. Within chloroplasts are found varying numbers of somewhat cylindrical structures called grana, which generally include fused stacks of flattened membranes called thylakoids. At or near the thylakoid membranes in grana, substantial portions of the photosynthetic process occur.
Extensive research into the process of biosynthesis of chlorophyll in lower and higher plants has been carried out, reported and reviewed within the past two decades. See, e.g., the references collected and cited by the inventor in, Rebeiz, et al., Ann. Rev. Plant. Physiol., 24, 129-72 at p. 131. The step-by-step enzymology of chlorophyll biosynthesis is still not completely known. The middle portion of the biosynthetic pathway, i.e., the stepwise conversion of .delta.-aminolevulinic acid (hereafter, ALA) to protoporphyrin IX, has been explored with moderate success in cell-free preparations from higher plants. However, the initial step involving ALA biosynthesis and the final steps involving conversion of protoporphyrin to chlorophyll are still largely unknown. Progress in these two areas has been held back by the lack of cell-free, in vitro, systems able to catalyze these reactions. The lack of such systems has been in major part due to the general inability to suppress in vitro, the "auto-destructive" activity of chloroplasts and related organelles upon their removal from in vivo systems.
In the last decade considerable efforts have been devoted to evaluating the reproductive, developmental and nutritional autonomy of chloroplasts. The control of chlorophyll biosynthesis and biosynthesis of thylakoid membranes have also received considerable attention. See, e.g., the reviews dealing with this topic collected and cited by the inventor in Rebeiz, et al., supra, at p. 132. Here, too, the progress of the field has been hindered by the lack of chloroplast preparations capable of doing in vitro, what they can be in vivo (see, e.g., Woodcock, et al., in "Structure and Function of Chloroplasts," (ed., Gibbs), pp. 89-128 (New York: Springer-Verlag, 1971)).
The inventor and his collaborators have shown that cell-free homogenates and etioplasts isolated from cucumber cotyledons and incubated with .sup.14 C-ALA and certain biosynthetic cofactors are capable of synthesizing .sup.14 C-Mg protoporphyrin monoester, .sup.14 C-Protochlorophyll and .sup.14 C-chlorophyll a and b. See, Rebeiz, et al., Plant Physiol., 47, 24-32 (1971); Rebeiz, et al., Plant Physiol., 47, 33-37 (1971); and Rebeiz, et al., Plant Physiol., 46, 543-49 (1970). Confirmation of that work was reported by Wellburn, et al., in Biochem. and Biophys, Res. Comm., 45, 747-50 (1971). (See also, Wellburn, et al., J. Experimental. Bot., 22, 972-79 (1971) and Wellburn, et al., J. Cell. Sci., 9, 271-87 (1971).)
The net biosynthesis of microgram quantities of chlorophyll from ALA in vitro has, however, heretofore not been achieved. Similarly, the biosynthetic in vitro assembly of grana has heretofore not been achieved.